PROJECT SUMMARY Craniofacial morphogenesis is a highly orchestrated and evolutionarily conserved developmental process. In some cases, changes to the genes that underlie normal craniofacial variation across vertebrates can cause anatomical defects in humans. Thus, a comprehensive understanding of the genetic and developmental mechanisms of craniofacial diversity will provide important insights into human congenital craniofacial disorders, which account for more than one third of all birth defects and often cause hearing loss, speech impediment and intellectual disability. Among breeds of domestic pigeon, radical variation in beak morphology within a single species has resulted from millennia of artificial selection. Preliminary genomic analyses support the results of classical breeding experiments, which suggest that pigeon beak morphology is controlled by multiple genetic loci, including at least one sex-linked factor. By comparing the genomes of small and medium beak pigeons, I have identified two candidate genes, ROR2 and SMAD6, which encode members of the non-canonical Wnt and BMP signaling pathways, respectively. In humans, mutations in these genes cause congenital craniofacial disorders, including Robinow syndrome and Craniosynostosis. This project utilizes cross-disciplinary approaches and complementary vertebrate models to understand the combinatorial function of specific genes and signaling pathways during patterning of the vertebrate craniofacial skeleton. Furthermore, this project takes advantage of the striking variation in beak morphology within domestic pigeons to identify the genetic architecture of specific dimensions of craniofacial morphology, including the size and shape of individual bones of the craniofacial skeleton. This project will pursue two distinct Aims. Aim 1 will test the individual and integrated molecular functions of ROR2 and SMAD6 during vertebrate craniofacial morphogenesis. I will use a combination of in situ hybridization (ISH) and RNA sequencing (RNA-seq) techniques to determine if differential expression of ROR2 and/or SMAD6 and their respective signaling pathways are associated with variation in beak morphology in avian embryos. In addition, I will employ genetic, developmental and live imaging approaches to test the functional roles of ROR2 and SMAD6 during chick and zebrafish craniofacial morphogenesis. Aim 2 will identify specific genes associated with variation in the size and shape of individual bones of the vertebrate craniofacial skeleton. I will perform quantitative trait locus (QTL) mapping in two pigeon F2 intercrosses to identify genomic loci that underlie variation in pigeon beak morphology. To complement the genetic mapping experiments, I will leverage the Shapiro lab's extensive ?pigeonomic? toolkit to fine map the specific genes or DNA sequences within each QTL that are associated with dimensions of beak morphology. These experiments will broadly define the genetic architecture of craniofacial variation in pigeons and will implicate specific candidate genes for further investigation. These complementary genetic, genomic, and developmental approaches will define the precise roles of two genes in vertebrate craniofacial morphogenesis and will identify the molecular basis of astonishing craniofacial variation in an innovative model system. Together, the Aims of this project will open new avenues to understand the roles of specific genes in normal and disease variation among vertebrates in general.